Innate Lymphoid Cells Subsets Research Articles

Investigating the impact of antibiotic-induced dysbiosis on protection from Clostridium difficile colitis by mouse colonic innate lymphoid cells.

Innate lymphoid cells (ILCs) play a critical role in maintaining intestinal health in homeostatic and diseased conditions. During Clostridium difficile infection (CDI), IL-33 activates ILC2 to protect from colonic damage and mortality. The function of IL-33 and ILC is tightly regulated by the intestinal microbiota. We set out to determine the impact of antibiotic-induced disruption of the microbiome on ILC function. Our goal was to understand antibiotic-induced changes in ILC function on susceptibility to C. difficile colitis in a mouse model. We utilized high-throughput single-cell RNAseq to investigate the phenotypic features of colonic ILC at baseline, after antibiotic administration with or without IL-33 treatment. We identified a heterogeneous landscape of colonic ILCs with gene signatures of inflammatory, anti-inflammatory, migratory, progenitor, plastic, and antigen-presenting ILCs. Antibiotic treatment decreased ILC2 while coordinately increasing ILC1 and ILC3 phenotypes. Notably, Ifng+, Ccl5+, and Il23r+ ILC increased after antibiotics. IL-33 treatment counteracted the antibiotic effect by downregulating ILC1 and ILC3 and activating ILC2. In addition, IL-33 treatment markedly induced the expression of type 2 genes, including Areg and Il5. Finally, we identified amphiregulin, produced by ILC2, as protective during C. difficile infection. Together, our data expand our understanding of how antibiotics induce susceptibility to C. difficile colitis through their impact on ILC subsets and function.IMPORTANCEClostridium difficile infection (CDI) accounts for around 500,000 symptomatic cases and over 20,000 deaths annually in the United States alone. A major risk factor of CDI is antibiotic-induced dysbiosis of the gut. Microbiota-regulated IL-33 and innate lymphoid cells (ILCs) are important in determining the outcomes of C. difficile infection. Understanding how antibiotic and IL-33 treatment alter the phenotype of colon ILCs is important to identify potential therapeutics. Here, we performed single-cell RNAseq of mouse colon ILCs collected at baseline, after antibiotic treatment, and after IL-33 treatment. We identified heterogeneous subpopulations of all three ILC subtypes in the mouse colon. Our analysis revealed several potential pathways of antibiotic-mediated increased susceptibility to intestinal infection. Our discovery that Areg is abundantly expressed by ILCs, and the protection of mice from CDI by amphiregulin treatment, suggests that the amphiregulin-epidermal growth factor receptor pathway is a potential therapeutic target for treating intestinal colitis.

Type 2 innate lymphoid cells are not involved in mouse bladder tumor development.

Therapies for bladder cancer patients are limited by side effects and failures, highlighting the need for novel targets to improve disease management. Given the emerging evidence highlighting the key role of innate lymphoid cell subsets, especially type 2 innate lymphoid cells (ILC2s), in shaping the tumor microenvironment and immune responses, we investigated the contribution of ILC2s in bladder tumor development. Using the orthotopic murine MB49 bladder tumor model, we found a strong enrichment of ILC2s in the bladder under steady-state conditions, comparable to that in the lung. However, as tumors grew, we observed an increase in ILC1s but no changes in ILC2s. Targeting ILC2s by blocking IL-4/IL-13 signaling pathways, IL-5, or IL-33 receptor, or using IL-33-deficient or ILC2-deficient mice, did not affect mice survival following bladder tumor implantation. Overall, these results suggest that ILC2s do not contribute significantly to bladder tumor development, yet further investigations are required to confirm these results in bladder cancer patients.

Clinical and basic science aspects of innate lymphoid cells as novel immunotherapeutic targets in cancer treatment.

Immunotherapy has revolutionised cancer treatment over the past decade, demonstrating remarkable efficacy across a broad range of cancer types. However, not all patients or cancer types respond to contemporary clinically-utilised immunotherapeutic strategies, which largely focus on harnessing adaptive immune T cells for cancer treatment. Accordingly, it is increasingly recognised that upstream innate immune pathways, which govern and orchestrate the downstream adaptive immune response, may prove critical in overcoming cancer immunotherapeutic resistance. Innate lymphoid cells (ILCs) are the most recently discovered major innate immune cell population. They have overarching roles in homeostasis and orchestrating protective immunity against pathogens. As innate immune counterparts of adaptive immune T cells, ILCs exert effector functions through the secretion of cytokines and direct cell-to-cell contact, with broad influence on the overall immune response. Importantly, dysregulation of ILC subsets have been associated with a range of diseases, including immunodeficiency disorders, allergy, autoimmunity, and more recently, cancer. ILCs may either promote or inhibit cancer initiation and progression depending on the cancer type and the specific ILC subsets involved. Critically, therapeutic targeting of ILCs and their associated cytokines shows promise against a wide range of cancer types in both preclinical models and early phase oncology clinical trials. This chapter provides a comprehensive overview of the current understanding of ILC subsets and the associated cytokines they produce in cancer pathogenesis, with specific focus on how these innate pathways are, or can be targeted, therapeutically to overcome therapeutic resistance and ultimately improve patient care.

Single cell multi-omic analysis identifies key genes differentially expressed in innate lymphoid cells from COVID-19 patients.

Innate lymphoid cells (ILCs) are enriched at mucosal surfaces where they respond rapidly to environmental stimuli and contribute to both tissue inflammation and healing. To gain insight into the role of ILCs in the pathology and recovery from COVID-19 infection, we employed a multi-omics approach consisting of Abseq and targeted mRNA sequencing to respectively probe the surface marker expression, transcriptional profile and heterogeneity of ILCs in peripheral blood of patients with COVID-19 compared with healthy controls. We found that the frequency of ILC1 and ILC2 cells was significantly increased in COVID-19 patients. Moreover, all ILC subsets displayed a significantly higher frequency of CD69-expressing cells, indicating a heightened state of activation. ILC2s from COVID-19 patients had the highest number of significantly differentially expressed (DE) genes. The most notable genes DE in COVID-19 vs healthy participants included a) genes associated with responses to virus infections and b) genes that support ILC self-proliferation, activation and homeostasis. In addition, differential gene regulatory network analysis revealed ILC-specific regulons and their interactions driving the differential gene expression in each ILC. Overall, this study provides mechanistic insights into the characteristics of ILC subsets activated during COVID-19 infection.

Disease pathogenesis and barrier functions regulated by group 3 innate lymphoid cells.

The mucosal surface is in constant contact with foreign antigens and is regulated by unique mechanisms that are different from immune responses in the peripheral organs. For the last several decades, only adaptive immune cells such as helper T (Th) cells, Th1, Th2, or Th17 were targeted to study a wide variety of immune responses in the mucosal tissues. However, since their discovery, innate lymphoid cells (ILCs) have been attracting attention as a unique subset of immune cells that provide border defense with various functions and tissue specificity. ILCs are classified into different groups based on cell differentiation and functions. Group 3 innate lymphoid cells (ILC3s) are particularly in close proximity to mucosal surfaces and therefore have the opportunity to be exposed to a variety of bacteria including pathogenic bacteria. In recent years, studies have also provided much evidence that ILC3s contribute to disease pathogenesis as well as the defense of mucosal surfaces by rapidly responding to pathogens and coordinating other immune cells. As the counterpart of helper T cells, ILC3s together with other ILC subsets establish the immune balance between adaptive and innate immunity in protecting us from invasion or encounter with non-self-antigens for maintaining a complex homeostasis. In this review, we summarize recent advances in our understanding of ILCs, with a particular focus on the function of ILC3s in their involvement in bacterial infection and disease pathogenesis.

Distinct regulatory machineries underlying divergent chromatin landscapes distinguish innate lymphoid cells from T helper cells.

Innate lymphoid cells (ILCs), as the innate counterpart of CD4+ T helper (Th) cells, play crucial roles in maintaining tissue homeostasis. While the ILC subsets and their corresponding Th subsets demonstrate significant similarities in core programming related to effector function and regulatory mechanisms, their principal distinctions, given their innate and adaptive lymphocyte nature, remain largely unknown. In this study, we have employed an integrative analysis of 294 bulk RNA-sequencing results across all ILC and Th subsets, using scRNA-seq algorithms. Consequently, we identify two genesets that predominantly differentiate ILCs from Th cells, as well as three genesets that distinguish various immune responses. Furthermore, through chromatin accessibility analysis, we find that the ILC geneset tends to rely on specific transcriptional regulation at promoter regions compared with the Th geneset. Additionally, we observe that ILCs and Th cells are under differential transcriptional regulation. For example, ILCs are under stronger regulation by multiple transcription factors, including RORα, GATA3, and NF-κB. Otherwise, Th cells are under stronger regulation by AP-1. Thus, our findings suggest that, despite the acknowledged similarities in effector functions between ILC subsets and corresponding Th subsets, the underlying regulatory machineries still exhibit substantial distinctions. These insights provide a comprehensive understanding of the unique roles played by each cell type during immune responses.

Abstract A009: CD103+CD56+ innate lymphoid cells are associated with an immunosuppressive microenvironment in ovarian cancer

Abstract Background: Regulatory innate lymphoid cells (ILCregs) are an emerging heterogenous subset of innate lymphoid cells that suppress immunity in several healthy and diseased contexts, including cancer. Previously, we identified CD56+ ILCregs from in vitro expanded tumour-infiltrating lymphocyte (TIL) cultures in patients with epithelial ovarian carcinoma (EOC). However, lineage-defining surface markers or transcription factors that distinguishes these regulatory populations from other conventional NK cells have not been identified. The objective of this study is to characterize markers that define ILCregs directly from primary tumours and investigate its role within the tumour microenvironment (TME). Methods: Women with suspected abdominal mass were recruited and consented at Gynecology Cancer Clinic at Princess Margaret Hospital. Surgically resected EOC tumours were processed and analyzed by flow cytometry and single-cell RNA-sequencing. Suppression assays were performed by co-culture of tumour-infiltrating CD103+CD56+ ILCregs with autologous TILs for 4 days in vitro with soluble αCD3. Results: We found that CD103 expressing CD56+ ILCregs were associated with poor clinical prognosis in patients with ovarian carcinoma. CD103+CD56+ ILCregs displayed reduced expression of cytolytic related markers (i.e., GZMB, CD107a, CD16) compared to CD103-CD56+ NK cells and had a distinct transcriptomic profile from NK cells. Interestingly, CD103+CD56+ ILCregs displayed distinct surface markers (i.e., CD49a, CD69, GITR) and transcription factor networks that were associated with Treg development, differentiation, and suppressive function. Intratumoural CD103+CD56+ ILCregs suppressed autologous CD8+ T cells by downregulation of granzyme B in vitro. Concordantly, the abundance of CD103+CD56+ ILCregs in patient tumours were associated with reduced activated CD8+ T cells within the TME. Conclusion: This study identified CD103+CD56+ ILCregs in the TME of patients with EOC that are associated with poor clinical prognosis and plays a key role in regulating T cells. Further investigations into underlying immunoregulatory networks, including ILCregs, within the TME may provide novel targets to improve prognosis for patients with EOC. Citation Format: Douglas C Chung, Jehan Vakharia, Kathrin Warner, Nicolas Jacquelot, Azin Sayad, SeongJun Han, Maryam Ghaedi, Carlos R Garcia-Batres, Alisha Elford, Ben X Wang, Linh T Nguyen, Patricia A Shaw, Blaise A Clarke, Marcus Q Bernardini, Sarah E Ferguson, Pamela S Ohashi. CD103+CD56+ innate lymphoid cells are associated with an immunosuppressive microenvironment in ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A009.

Type 3 Innate Lymphoid Cells Promote Acute Myeloid Leukemia Immune Evasion and Expansion

Acute Myeloid Leukemia (AML) is an aggressive myeloid malignancy originated in the bone marrow with poor overall survival rates of less than 30%. The immune system has been shown to play an important role in detecting and eliminating leukemic cells. Recent studies have described a population of immune cells termed Innate lymphoid cells (ILCs). ILCs are known to regulate both innate and adaptive immunity in an antigen-independent manner and are believed to be among the first immune cells to interact with malignantly transformed cells. Natural killer (NK) cells are a subset of ILCs and are known to inhibit AML progression, however the impact of other ILCs is not yet fully understood. Type 3 ILCs (ILC3s) have key roles in maintaining homeostasis in mucosal immunity through their ability to secrete cytokines including IL-17, IL-22, TNFα and GM-CSF (pro-myeloid). An expansion of ILC3s has recently been observed in solid tumors, however the overall functional impact of ILC3s in the setting of AML remains poorly understood and warrants further investigation. We first utilized an MLL-partial tandem duplication and FLT3-internal tandem duplication Flt3-ITD double knock-in mutation murine model of AML that recapitulates two clinically relevant mutations in human AML. Using this AML model, we discovered an expansion of ILC3s in the bone marrow of leukemic mice compared to wild-type control mice (2.364% +/- 0.40% AML vs 0.045% +/- 0.40% WT, n=5, p<0.0001). We also observed an increase in ILC3s in the bone marrow of untreated AML patients relative to age matched donor controls (7.013% +/- 2.6% vs. 2.328% +/- 2.6%). Our published data have previously shown AML blasts secrete aryl hydrocarbon receptor (AHR) ligands, which can bind and activate the transcription factor AHR in ILC precursors and mature ILCs. AHR is an important transcription factor in ILC3 development. Therefore, we hypothesized that the ILC3 increase was due at least in part to AHR-mediated increased ILC3 differentiation. To address this hypothesis, we isolated the ILC precursor (ILCP) from normal human donors and co-cultured with AML cells. By regulating AHR activity in this system, we showed that ILC3s are expanded in AML in part due to AML-mediated AHR activation (28.02% +/- 3.89% without AML, 60% +/- 5.5% with AML and 27% +/- 5% AML+AHR inhibitor, n=15, p<0.001). Next, we sought to determine the direct impact of ILC3s on AML proliferation and differentiation that could contribute to AML relapse. We utilized a colony forming assay (CFU) assay in the presence or absence of ILC3s. Bone marrow from primary untreated AML patients was cultured alone or with ILC3s (Lin-CD94-NKp44+). AML samples co-cultured with ILC3s had significantly increased colony formation relative to AML alone (69.4 CFU AML+ILC3 vs 42.7 CFU AML alone, n=8, p<0.05). Post-culture, we observed significant increases in the transcript of GM-CSF and TNFα in ILC3s co-cultured with AML marrow as compared to ILC3s alone. Furthermore, in our immunocompetent murine AML model, we observed significantly higher production of GM-CSF and TNFα by ILC3s in the bone marrow of leukemic mice relative to nonleukemic controls (GM-CSF: 11.08% +/- 1.6 AML vs 1.128% +/- 1.6 WT, n=10, p<0.0001; TNFα: 5.456% +/- 1.3 AML vs 1.307% +/- 1.3 WT, n=10, p<0.05), suggesting a potential mechanism of ILC3-mediated increased colony formation. Lastly, to determine if ILC3 expansion impacted NK cell immune surveillance, we co-cultured normal donor human ILC3s with AML cells, separated by transwell, for 48 hours before subjecting the AML cells to an NK cell cytotoxicity assay. We observed significantly reduced NK cell killing of AML cells when co-cultured with ILC3s compared to controls, suggesting ILC3s protect AML cells from NK cell-mediated killing (24.3% killing +/- 3.81% AML alone vs 14.57% +/- 1.35% AML with ILC3, n=18, p<0.01). Collectively, these data support a model in which AML-mediated AHR activation promotes ILC3 expansion to both support AML proliferation and protect AML blasts from immune surveillance. Future studies will focus in exploring how to target these interactions to design effective therapeutic approaches and improve patient clinical outcomes.

Both Horatio and Polonius: Innate Lymphoid Cells in Tissue Homeostasis and Repair.

Innate lymphoid cells (ILCs) have emerged as critical tissue-resident lymphocytes that coordinate responses to environmental stress and injury. Traditionally, their function was thought to mirror adaptive lymphocytes that respond to specific pathogens. However, recent work has uncovered a more central role for ILCs in maintaining homeostasis even in the absence of infection. ILCs are now better conceptualized as an environmental rheostat that helps maintain the local tissue setpoint during environmental challenge by integrating sensory stimuli to direct homeostatic barrier and repair programs. In this article, we trace the developmental origins of ILCs, relate how ILCs sense danger signals, and describe their subsequent engagement of appropriate repair responses using a general paradigm of ILCs functioning as central controllers in tissue circuits. We propose that these interactions form the basis for how ILC subsets maintain organ function and organismal homeostasis, with important implications for human health.

Crosstalk between ESKAPE bacteria and NK cells: mutual regulation and role in developing reproductive tract pathologies

Natural killer (NK) cells represent one of the innate lymphoid cell subsets, which are often studied in the context of antitumor and antiviral immunity, as well as due to their localization in the zone of the mother-fetus contact (in the uterus), therefore underlying their extensive investigation in developing pregnancy. At the same time, their role in antibacterial immune response has been poorly examined. Because NK cells can produce cytokines, one of putative options for their participation in eliminating prokaryotic pathogens may be coupled to regulation of immune system cells such as dendritic cells, macrophages, etc. However, there have been also described variants of contact cytolysis of cells infected with intracellular bacteria enabled due to cytotoxic proteins perforin, granzymes, granulisin found in NK cells. In recent years, it has become known that NK cells take part in development of immune response against extracellular bacteria including the ESKAPE group bacteria, which includes opportunistic prokaryotes that most actively develop antibiotic resistance and cause nosocomial infections. Here, we attempted to review the data on the role NK cells play in antibacterial immunity. Assessing a crosstalk between ESKAPE group bacteria and NK cells also attracts researchers due to the ability of prokaryotes to alter functions of immune cells, but very little is known about the effects they exert on NK cells. At the same time, such data could be applied to seek out for new ways to treat oncological diseases as well as pave the basis for new approaches to regulating NK cell characteristics in reproductive pathologies. As mentioned earlier, the latter occur in the decidual membrane, where they can interact with fetal cells including trophoblast cells. It is believed that cells can mutually regulate each others properties necessary for the course of physiological pregnancy. Probably, imbalance in this system can lead to development of reproductive pathologies. The review summarizes the currently available data on the effects of ESKAPE group bacteria on NK cells, and also considers putative mechanisms for emergence of impaired interaction between NK cells and trophoblasts exposed to ESKAPE group bacteria. Owing to few publications available on this phenomenon, the experimental study assessing an impact of ESKAPE group bacteria on NK cell properties is envisioned as a necessary stage in development of contemporary biology.

Proline uptake promotes activation of lymphoid tissue inducer cells to maintain gut homeostasis.

Metabolic regulation is integral to the proper functioning of innate lymphoid cells, yet the underlying mechanisms remain elusive. Here, we show that disruption of exogenous proline uptake, either through dietary restriction or by deficiency of the proline transporter Slc6a7, in lymphoid tissue inducer (LTi) cells, impairs LTi activation and aggravates dextran sodium sulfate-induced colitis in mice. With an integrative transcriptomic and metabolomic analysis, we profile the metabolic characteristics of various innate lymphoid cell subsets and reveal a notable enrichment of proline metabolism in LTi cells. Mechanistically, defective proline uptake diminishes the generation of reactive oxygen species, previously known to facilitate LTi activation. Additionally, LTi cells deficient in Slc6a7 display downregulation of Cebpb and Kdm6b, resulting in compromised transcriptional and epigenetic regulation of interleukin-22. Furthermore, our study uncovers the therapeutic potential of proline supplementation in alleviating colitis. Therefore, these findings shed light on the role of proline in facilitating LTi activation and ultimately contributing to gut homeostasis.

The importance of alterations in innate lymphoid cell subsets in patients with non-small cell lung cancer and their role in tumorigenesis

Objective. Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related morbidity and mortality. Diverse functions of innate lymphoid cells (ILCs) and NK cell subsets are investigated thoroughly in cancer immunotherapy. ILC and recently described NK cell subsets in NSCLC patients’ blood samples and tumor draining lymph nodes were investigated.
 Methods. The study included chemotherapy and/or radiotherapy-naive NSCLC patients with clinical stage T1-4N0-2M0 who underwent video-assisted mediastinal lymphadenectomy and 14 healthy controls. Mononuclear cells were isolated from peripheral blood of both groups and mediastinal lymph nodes of NSCLC patients. NK cells and ILC subsets were analyzed by flow cytometry.
 Results. Total NK cells are shown to be increased in peripheral blood of NSCLC patients compared to lymph nodes while the ratio of CD56dimCD16- exhausted NK cells is higher in lymph nodes than in blood samples of NSCLC patients. Compared to control group, peripheral blood ILC1 cells were lower in NSCLC patients, however ILC2 and ILC3 cells were significantly increased. However, mediastinal lymph nodes of NSCLC patients had decreased ratio of ILC2 and increased ratio of ILC3 cells than in peripheral blood of patients. NSCLC patients had significantly increased ratio of NKp44-ILC3 cells and decreased ratio of NKp44+ILC3 in lymph nodes. 
 Conclusion. Decreased ratio of ILC1 cells is an important indicator of impaired anti-tumoral response. Increased in the ratio of NKp44-ILC3 cells in NSCLC patients may potentially contribute to tumor progression. These findings highlight the distinct roles of ILCs, which play a pivotal role in the pathogenesis of lung cancer.

In-Depth Proteomic Map of Innate Lymphoid Cells from Healthy Human Skin and Blood

Innate lymphoid cells (ILCs) are essential players in the skin-associated immune system, nevertheless little is known about their proteomes and proteomic diversity. In this study, we describe about 6,600 proteins constitutively expressed by ILC2s and ILC3s from healthy human skin and blood using state-of-the-art proteomics. Although the vast majority of proteins was expressed by both ILC subsets and in both compartments, the skin ILC2s and ILC3s were more distinct than their counterparts in blood. Only skin ILC3s expressed uniquely detected proteins. Our in-depth proteomic datasetallowed us to define the cluster of differentiation marker profiles of the ILC subsets, explore distribution and abundance of proteins known to have immunological functions, as well as identify subset-specific proteins that have not previously been implicated in ILC biology. Taken together, our analyses substantially expand understanding of the protein expression signatures of ILC subsets. Going forward, these proteomic datasets will serve as valuable resources for future studies of ILC biology.

Transcription factor TCF-1 regulates the functions, but not the development, of lymphoid tissue inducer subsets in different tissues

Lymphoid tissue inducer (LTi) cells, a subset of innate lymphoid cells (ILCs), play an essential role in the formation of secondary lymphoid tissues. However, the regulation of the development and functions of this ILC subset is still elusive. In this study, we report that the transcription factor Tcell factor 1 (TCF-1), just as GATA3, is indispensable for the development of non-LTi ILC subsets. While LTi cells are still present in TCF-1-deficient mice, the organogenesis of Peyer's patches (PPs), but not of lymph nodes, is impaired in these mice. LTi cells from different tissues have distinct gene expression patterns, and TCF-1 regulates the expression of lymphotoxin specifically in PP LTi cells. Mechanistically, TCF-1 may directly and/or indirectly regulate Lta, including through promoting the expression of GATA3. Thus, the TCF-1-GATA3 axis, which plays an important role during Tcell development, also critically regulates the development of non-LTi cells and tissue-specific functions of LTi cells.

Transcriptional and chromatin profiling of human blood innate lymphoid cell subsets sheds light on HIV‐1 pathogenesis

Innate lymphoid cells (ILCs) are a diverse population of cells that include NK cells and contribute to tissue homeostasis and repair, inflammation, and provide protection from infection. The interplay between human blood ILCs, as well as their responses to HIV‐1 infection, remains poorly understood. This study used transcriptional and chromatin profiling to explore these questions. Transcriptional profiling and flow cytometry analysis support that there are four main ILC subsets found in human blood. Unlike in mice, human NK cells expressed the tissue repair protein amphiregulin (AREG). AREG production was induced by TCF7/WNT, IL‐2, and IL‐15, and inhibited by TGFB1, a cytokine increased in people living with HIV‐1. In HIV‐1 infection, the percentage of AREG+ NK cells correlated positively with the numbers of ILCs and CD4+ T cells but negatively with the concentration of inflammatory cytokine IL‐6. NK‐cell knockout of the TGFB1‐stimulated WNT antagonist RUNX3 increased AREG production. Antiviral gene expression was increased in all ILC subsets from HIV‐1 viremic people, and anti‐inflammatory gene MYDGF was increased in an NK‐cell subset from HIV‐1‐infected people whose viral load was undetectable in the absence of antiretroviral therapy. The percentage of defective NK cells in people living with HIV‐1 correlated inversely with ILC percentage and CD4+ T‐cell counts. CD4+ T cells and their production of IL‐2 prevented the loss of NK‐cell function by activating mTOR. These studies clarify how ILC subsets are interrelated and provide insight into how HIV‐1 infection disrupts NK cells, including an uncharacterized homeostatic function in NK cells.

A Blinded Investigation: Accentuated NK Lymphocyte CD335 (NKp46) Expression Predicts Pregnancy Failures.

NKp46 is an NK cell receptor uniquely expressed by NK cells and a small subset of innate lymphoid cells. In our previous studies, we suggested a tight connection between the activity of NK cells and the expression of NKp46 and supported the clinical significance of NKp46 expression in NK cells in women with reproductive failures. In this study, we investigated the expression of NKp46 in NK cells in the peripheral blood of women in early pregnancy and analyzed its association with pregnancy loss. In a blinded study, we examined blood samples and analyzed the subsequent pregnancy outcomes from 98 early pregnant women (5th-7th week of gestation-w.g.) and 66 women in the 11th-13th week of pregnancy who served as controls. We studied the expression of NKp46 and the levels of anti-cardiolipin antibodies (aCL). The results of aCL were shared with the clinic, while the expression of NKp46 was blinded and not analyzed until the end of the study. A misbalance in the NKp46+NK cells subpopulations was associated with an unfavorable ongoing pregnancy. A decreased level of NKp46high cells (<14%) was strongly associated with miscarriage. A decreased level of the double-bright subpopulation (NKp46hightCD56++) also was a negative prognostic factor for the pregnancy course, but its increased level (>4%) was strongly associated with a successful pregnancy course. Our results showed that accentuated levels of NKp46+NK cells lead to a negative prognosis for early pregnancy courses in women.

Single-cell landscape dissecting the transcription and heterogeneity of innate lymphoid cells in ischemic heart.

Until now, few articles have revealed the potential roles of innate lymphoid cells (ILCs) in cardiovascular diseases. However, the infiltration of ILC subsets in ischemic myocardium, the roles of ILC subsets in myocardial infarction (MI) and myocardial ischemia-reperfusion injury (MIRI) and the related cellular and molecular mechanisms have not been described with a sufficient level of detail. In the current study, 8-week-old male C57BL/6J mice were divided into three groups: MI, MIRI and sham group. Single-cell sequencing technology was used to perform dimensionality reduction clustering of ILC to analyze the ILC subset landscape at a single-cell resolution, and finally flow cytometry was used to confirm the existence of the new ILC subsets in different disease groups. Five ILC subsets were found, including ILC1, ILC2a, ILC2b, ILCdc and ILCt. It is worth noting that ILCdc, ILC2b and ILCt were identified as new ILC subclusters in the heart. The cellular landscapes of ILCs were revealed and signal pathways were predicted. Furthermore, pseudotime trajectory analysis exhibited different ILC statuses and traced related gene expression in normal and ischemic conditions. In addition, we established a ligand-receptor-transcription factor-target gene regulatory network to disclose cell communications among ILC clusters. Moreover, we further revealed the transcriptional features of the ILCdc and ILC2a subsets. Finally, the existence of ILCdc was confirmed by flow cytometry. Collectively, by characterizing the spectrums of ILC subclusters, our results provide a new blueprint for understanding ILC subclusters' roles in myocardial ischemia diseases and further potential treatment targets.

Programmed cell death-1 receptor-mediated regulation of Tbet+NK1.1− innate lymphoid cells within the tumor microenvironment

Innate lymphoid cells (ILCs) play a key role in tissue-mediated immunity and can be controlled by coreceptor signaling. Here, we define a subset of ILCs that are Tbet+NK1.1- and are present within the tumor microenvironment (TME). We show programmed death-1 receptor (PD-1) expression on ILCs within TME is found in Tbet+NK1.1- ILCs. PD-1 significantly controlled the proliferation and function of Tbet+NK1.1- ILCs in multiple murine and human tumors. We found tumor-derived lactate enhanced PD-1 expression on Tbet+NK1.1- ILCs within the TME, which resulted in dampened the mammalian target of rapamycin (mTOR) signaling along with increased fatty acid uptake. In line with these metabolic changes, PD-1-deficient Tbet+NK1.1- ILCs expressed significantly increased IFNγ and granzyme B and K. Furthermore, PD-1-deficient Tbet+NK1.1- ILCs contributed toward diminished tumor growth in an experimental murine model of melanoma. These data demonstrate that PD-1 can regulate antitumor responses of Tbet+NK1.1- ILCs within the TME.

Plasticity between type 2 innate lymphoid cell subsets and amphiregulin expression regulates epithelial repair in biliary atresia.

Although a dysregulated type 1 immune response is integral to the pathogenesis of biliary atresia, studies in both humans and mice have uncovered a type 2 response, primarily driven by type 2 innate lymphoid cells. In nonhepatic tissues, natural type 2 innate lymphoid cell (nILC2s) regulate epithelial proliferation and tissue repair, whereas inflammatory ILC2s (iIlC2s) drive tissue inflammation and injury. The aim of this study is to determine the mechanisms used by type 2 innate lymphoid cell (ILC2) subpopulations to regulate biliary epithelial response to an injury. Using Spearman correlation analysis, nILC2 transcripts, but not those of iILC2s, are positively associated with cholangiocyte abundance in biliary atresia patients at the time of diagnosis. nILC2s are identified in the mouse liver through flow cytometry. They undergo expansion and increase amphiregulin production after IL-33 administration. This drives epithelial proliferation dependent on the IL-13/IL-4Rα/STAT6 pathway as determined by decreased nILC2s and reduced epithelial proliferation in knockout strains. The addition of IL-2 promotes inter-lineage plasticity towards a nILC2 phenotype. In experimental biliary atresia induced by rotavirus, this pathway promotes epithelial repair and tissue regeneration. The genetic loss or molecular inhibition of any part of this circuit switches nILC2s to inflammatory type 2 innate lymphoid cell-like, resulting in decreased amphiregulin production, decreased epithelial proliferation, and the full phenotype of experimental biliary atresia. These findings identify a key function of the IL-13/IL-4Rα/STAT6 pathway in ILC2 plasticity and an alternate circuit driven by IL-2 to promote nILC2 stability and amphiregulin expression. This pathway induces epithelial homeostasis and repair in experimental biliary atresia.

Innate lymphoid cells and innate-like T cells in cancer- at the crossroads of innate and adaptive immunity.

Immunotherapies targeting conventional T cells have revolutionized systemic treatment for many cancers, yet only a subset of patients benefit from these approaches. A better understanding of the complex immune microenvironment of tumours is needed to design the next generation of immunotherapeutics. Innate lymphoid cells (ILCs) and innate-like T cells (ILTCs) are abundant, tissue-resident lymphocytes that have recently been shown to have critical roles in many types of cancers. ILCs and ILTCs rapidly respond to changes in their surrounding environment and act as the first responders to bridge innate and adaptive immunity. This places ILCs and ILTCs as pivotal orchestrators of the final antitumour immune response. In this Review, we outline hallmarks of ILCs and ILTCs and discuss their emerging role in antitumour immunity, as well as the pathophysiological adaptations leading to their pro-tumorigenic function. We explore the pleiotropic, in parts redundant and sometimes opposing, mechanisms that underlie the delicate interplay between the different subsets of ILCs and ILTCs. Finally, we highlight their role in amplifying and complementing conventional T cell functions and summarize immunotherapeutic strategies for targeting ILCs and ILTCs in cancer.